Influenza A virus is a negative strand RNA virus belonging to the orthomyxovirus family. The genome of the virus consists of 8 segments and encodes 10 polypeptides. Experimental evidence generated in the laboratory of Scholtissek indicates that the nucleoprotein (NP) is a major determinant of species specificity of influenza viruses (Scholtissek, et al., 1985, Virology 147: 287-294). Phylogenetic analysis divides NP genes into two families: one containing NPs predominantly of avian origin, and one containing those of human origin (Bean, 1984, Virology 133:438-442; Buckler-White & Murphy, 1986, Virology 155: 345-355; Gammelin, et al., 1989, Virology 170:71-80; Scholtissek, et al., 1985, supra). The human virus A/HK/1/68 and viruses having genetically related NPs cannot rescue mutants of the avian virus A/FPV/Rostock/1/34 with ts defects in the NP following double infection of chicken embryo fibroblasts (CEF) at 40° C. (Scholtissek, et al., 1985, supra; Scholtissek, et al., 1978, Virology 91: 79-85). However, the human viruses which failed to rescue the ts mutants on CEF cells were able to do so on Madin-Darby canine kidney(MDCK) cells (Scholtissek, et al., 1978, supra). Additionally, A/HK/1/68 virus and A/FPV/Rostock/1/34 virus reassortants containing the A/HK/1/68 virus-derived NP replicate in MDBK cells but not in CEFs (Scholtissek, et al., 1978, supra). The host-specific rescue of FPV ts mutants and the host restriction of A/HK/1/68 virus reassortants suggest that a factor(s) of host origin, which differs between mammalian and avian cells, is responsible for this phenomenon, and that this factor may interact with the influenza A virus NP. However, heretofore, no host protein has been identified.
Replication and transcription of influenza virus RNA requires four virus encoded proteins: the NP and the three components of the viral RNA-dependent RNA polymerase, PB1, PB2 and PA (Huang, et al., 1990, J. Virol. 64: 5669-5673). The NP is the major structural component of the virion which interacts with genomic RNA, and is required for antitermination during RNA synthesis (Beaton & Krug, 1986, Proc. Natl. Acad. Sci. USA 83:6282-6286). NP is also required for elongation of RNA chains (Shapiro & Krug, 1988, J. Virol. 62: 2285-2290) but not for initiation (Honda, et al., 1988, J. Biochem. 104: 1021-1026).
NS1 is a major non-structural protein expressed by influenza A viruses in infected cells, whose role in infection is not clear. Studies of viruses carrying temperature-sensitive NS1 alleles point to a regulatory role for NS1 in viral gene-expression and/or replication (Wolstenholme, et al., 1980, J. Virol. 35:1-7; Koennecke, et al., 1981, Virol. 110:16-25; Hatada, et al., 1990, J. Gen. Virol. 71: 1283-1292), which is also consistent with its preferentially nuclear accumulation (Greenspan, et al., 1988, J. Virol. 62: 3020-3026). Its expression has been shown to interfere with cellular functions in a variety of ways. (Fortes, et al., 1994, EMBO J. 13: 704-712; Qiu & Krug, 1994, J. Virol. 68: 2425-2432; Lu, et al., 1994, Genes Dev. 8: 1817-1828). These effects have been suggested to be mediated through NS1's observed interactions with a variety of RNA's, including single- and double-stranded influenza vRNA (Hatada & Fukuda, 1992, J. Gen. Virol. 73: 3325-3329; Hatada, et al., 1992, J. Gen Virol. 73: 17-25), poly-adenosine RNA (Qiu & Krug, 1994, supra), and spliceosomal U6 RNA (Lu, et al., 1994, supra). Despite these studies involving the interaction of NS1 with various RNAs, no host proteins that interact with NS1 during infection have previously been identified or characterized.
Little is known about host cell functions which contribute to the intracellular replication of influenza viruses, and cellular factors have not been characterized which directly interact with the viral proteins, much less cellular factor/viral interactions that can be used as targets for therapeutic intervention.